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. 1997 Feb 18;94(4):1402-7.
doi: 10.1073/pnas.94.4.1402.

Intestinal tumorigenesis is suppressed in mice lacking the metalloproteinase matrilysin

C L Wilson  1 , K J Heppner, P A Labosky, B L Hogan, L M Matrisian
Affiliations

Intestinal tumorigenesis is suppressed in mice lacking the metalloproteinase matrilysin

C L Wilson et al. Proc Natl Acad Sci U S A. .

Abstract

Matrix metalloproteinases (MMPs) classically have been implicated in basement membrane destruction associated with late-stage tumor cell invasion and metastasis. However, recent studies have demonstrated that one MMP family member, matrilysin, is expressed in a high percentage of early-stage human colorectal tumors. We analyzed matrilysin expression in benign intestinal tumors from mice heterozygous for the ApcMin allele (Min/+) and found that the mRNA was induced in the majority (88%) of these adenomas. Protein was detected in the tumor cells, where, surprisingly, it was predominantly immunolocalized to the lumenal surface of dysplastic glands rather than the basement membrane or extracellular matrix. To address the role of matrilysin in Min intestinal tumorigenesis, we generated Min/+ mice deficient in this MMP by gene targeting and homologous recombination. The absence of matrilysin resulted in a reduction in mean tumor multiplicity in Min/+ animals of approximately 60% and a significant decrease in the average tumor diameter. Based on these findings, we conclude that matrilysin is a suppressor of the Min phenotype, possibly by functioning in a capacity independent of matrix degradation. These results argue for the use of MMP inhibitors in the treatment and prevention of early-stage colon cancer.

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Figures

Figure 1
Figure 1
Localization of matrilysin and gelatinase A in Min/+ tumors. In situ hybridization of matrilysin (A and B) and gelatinase A (C and D). Photomicrographs were taken using a double exposure with a red filter on dark field illumination so that the silver grains appear pink. (A) In a Min colonic adenoma, matrilysin mRNA is expressed by tumor (T) epithelium but not normal (N) colonic mucosa. (×125.) (B) Matrilysin mRNA localized to the dysplastic epithelium of a small intestinal tumor (arrow) and is absent in normal intestinal glandular epithelium. (×400.) (C) Gelatinase A mRNA is expressed in the tumor (T) compared with the adjacent normal (N) mucosa in a serial section of the colonic tumor shown in A. (×125.) (D) Gelatinase A mRNA localizes within the stroma (arrow) of a colonic tumor. (×250.) Immunolocalization of matrilysin protein (E and F). Matrilysin protein was visualized with TrueBlue color substrate (dark blue to purple), and nuclei were counterstained with Contrast Red. (E) Sporadic localization of matrilysin protein in a small intestinal tumor (T). (×160.) (F) Matrilysin immunoreactivity is frequently observed within the lumen of glandular structures (arrow). (×640.)
Figure 2
Figure 2
Mapping and targeted disruption of the matrilysin gene. (A) The BSS backcross panel [(C57BL/6JEi × SPRET/Ei)F1 × SPRET/Ei] from The Jackson Laboratory Backcross DNA Panel Map Service (19) was used to map the matrilysin locus (Mmp7). The segregation patterns of Mmp7 and several linked loci (D9Bir4, Ppib-rs1, and D9Mit160) among the backcross offspring are depicted by the columns of boxes, where each column represents the chromosome inherited from the (C57BL/6JEi × SPRET/Ei)F1 parent. C57BL/6JEi and SPRET/Ei alleles are indicated by the filled and open boxes, respectively. The number at the bottom of each column is the number of offspring with that haplotype. Recombination distances in cM (R) and standard errors (SE) are indicated to the right of the columns. The Chromosome Committee map places the metalloelastase locus (Mmel) at the same offset as D9Bir4. D9Bir3 may be the only mapped locus more proximal than Mmp7. (B) Structure of genomic segment, targeting vector, and disrupted allele. The top diagram depicts the 6.5-kb BamHI (B) genomic fragment containing exons 2–6 (E2-E6; hatched boxes) that was used to generate the targeting construct. Introns, extragenic sequence, and plasmid sequence are denoted by the heavy, thin, and wavy lines, respectively. As shown in the middle diagram, the phosphoglycerate kinase–neomycin cassette containing the phosphoglycerate kinase promoter (filled box), neomycin phosphotransferase cDNA (Neo), and a polyadenylylation signal (+) was inserted using the EcoRV (RV) and StuI sites (S) indicated. The herpes simplex virus-thymidine kinase (TK) cassette, composed of the TK promoter (barred box) and a polyadenylylation signal (+), was used for negative selection in the presence of gancyclovir. The construct was linearized at the unique NotI (N) site. The bottom diagram shows the expected structure of the targeted allele following electroporation into R1 ES cells and selection for doubly resistant colonies. The locations of the BamHI and BstEII (Bs) restriction sites and fragments (probes A and B) used for Southern blotting are also indicated. (C) Southern analysis of F2 progeny from heterozygote matings. Tail DNA was digested with BstEII and Southern blotted using probe A, which detects bands of 6.3 and 5.3 kb corresponding to the targeted (−) and wild-type (+) alleles, respectively. (D) Northern blot analysis of small intestinal RNA. Total RNA was extracted from the entire small intestine of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) F2 animals and was subjected to Northern blotting using a 3′ matrilysin cDNA probe as described (MMAT2; ref. 5). As a loading control, the blot was also probed with radiolabeled cryptdin-1 (CRP-1) cDNA, which detects an abundant message of approximately 450–480 bp in the small intestine (26).
Figure 3
Figure 3
Effect of the matrilysin null mutation on tumor multiplicity and diameter in Min/+ animals. Min/+; mmp7−/− N5F1 (>96% C57BL/6) mice were generated as described in Materials and Methods. These mice were analyzed in comparison to Min/+ animals for tumor number (A) and diameter (B). The mean and median numbers of tumors for Min/+ were 25.4 and 24 (range = 20–38, n = 14), respectively, and for Min/+; mmp7−/−, the numbers were 10.5 and 11 (range = 7 to 12, n = 6), respectively (P < 0.0006). The mean diameters of tumors per mouse for Min/+ and Min/+; mmp7−/− were 2.0 and 1.6 mm, respectively (P < 0.003). The nonparametric Wilcoxon Rank Sum test was used to analyze the statistical significance of all data.
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